Hemophilia is an inherited disease which has been known for centuries, but it is only within the last four decades that it has been possible to differentiate between the various forms; hemophilia A and hemophilia B. Hemophilia A is the most frequent form. It affects only males with an incidence of one or two individuals per 10 000 live-born males. The disease is caused by strongly decreased level or absence of biologically active coagulation factor VIII (antihemophilic factor), which is a protein normally present in plasma. The clinical manifestation of hemophilia A is a strong bleeding tendency and before treatment with factor VIII concentrates was introduced, the mean age of the patients concerned was less than 20 years. Concentrates of factor VIII obtained from plasma have been available for about three decades. This has improved the situation for treatment of hemophilia patients considerably and offered them the possibility of living a normal life.
Until recently, therapeutic factor VIII concentrates have been prepared by fractionation of plasma. However, there are since some years methods available for production of factor VIII in cell culture using recombinant DNA techniques as reported in e.g. W. Wood et al, Nature, 312, p. 330-37 (1984) and EP-A-0 160 457.
Factor VIII concentrates derived from human plasma contain several fragmented fully active factor VIII forms as described by Andersson et al, Proc. Natl. Acad. Sci. USA, 83, p. 2979-83 (May 1986). The smallest active form has a molecular mass of 170 kDa and consists of two chains of 90 kDa and 80 kDa held together by metal ion(s). Reference is here made to EP-A-0 197 901.
Pharmacia AB of Stockholm, Sweden, has developed a recombinant factor VIII product which corresponds to the 170 kDa plasma factor VIII form in therapeutic factor VIII concentrates. The truncated recombinant factor VIII molecule is termed r-VIII SQ and is produced by Chinese Hamster Ovary (CHO) cells in a cell culture process in serum-free medium.
The structure and biochemistry of recombinant factor VIII products in geneal have been described by Kaufman in Trends in Biotechnology, 9 (1991) and Hematology, 63, p. 155-65 (1991). The structure and biochemistry of r-VIII SQ have been described in WO-A-9109122.
High-performance hydrophobic interaction chromatography (HIC) is a separation technique suitable for purifying proteins. The general characteristics and suitable conditions for carrying out a HIC step have been described by K-O Eriksson in Protein Purification; Principles, High Resolution Methods, and Applications, VCH Publishers, Inc., New York, p. 207-226 (1989). In this technique, proteins are eluted from relatively weak hydrophobic stationary phases using a solution with decreasing ionic strength, introducing a surfactant, changing the polarity of the solvent and/or simply lowering the temperature. Such relatively mild conditions favor the recovery of proteins with essentially retained activity.
The factors affecting adsorption and desorption in HIC are thoroughly reviewed by T. Arakawa and L. Owers Narhi in Biotechnol. Appl. Biochem., 13, p. 151-172 (1991). The influence of surfactants on the interaction of various proteins with a HIC gel (resin) has been described by J. J. Buckley and D. B. Wetlaufer, J. Chrom., 518, p. 99-110 (1990). In this reference, however, the main purpose was to evaluate the influence of the surfactants on the gradient elution profiles.
Various gels (resins) exposing hydrophobic or semihydrophobic ligands have been described, for instance agaroses derivatized with aminoalkyl or diaminoalkyl groups. In the prior art, there are examples of purification of factor VIII by utilizing such gels. For instance, butyl-agarose gel was prepared by derivatizing Sepharose.RTM. 4B (sold by Pharmacia AB of Uppsala, Sweden) with butyl-amine, using CNBr-coupling technique. The resulting gel was utilized for purification of factor VIII, as reported by Th. Vukovich et al in Folia Haematol. Leipzig, 107 (1), p. 148-151 (1979) and Th. Vukovich et al in Haemostasis and Thrombosis (Proc. Serono Symp.), G. G. Neri Semeri and C. R. M. Prentice eds., 15, p. 407-410 (1979). A protein mixture was adsorbed onto this gel at very low ionic strength. Desorption was accomplished by increasing the concentration of sodium chloride. This clearly indicates that the forces utilized were predominantly electrostatic, i.e. mechanisms involved were those normally referred to as ion-exchange chromatography (IEC). Furthermore, it has been reported, for instance by B.-L. Johansson and I. Drevin in J. Chrom., 321, p. 335-342 (1985), that the CNBr-coupling technique creates an iso-urea group between the ligand and the matrix, which is positively charged at acid and neutral pH. This profoundly influences the overall properties of the gel.
Morgenthaler has compared a series of agaroses derivatized with aminoalkyl- or diaminoalkyl groups for purification of factor VIII, as described in Thromb. Haemostas., 47(2), p. 124 (1982). It was found, that it was difficult to attain a reversible binding of factor VIII to the agarose gels derivatized with aminoalkyl groups (alkane Sepharose.RTM.). Thus, salt induced elution was attained only to some extent and only from gels derivatized with short aminoalkyl groups using CNBr-coupling technique. Ethylene glycol elution failed independently of the length of the alkyl chain. In contrast, it has been found, that by addition of detergents at a concentration of .gtoreq.0.1%, desoiption of factor VIII from such columns could be achieved (EP-A-0 209 041). In the examples shown, the capacity of the HIC columns and the concentration of the eluted factor were however low.
EP-A-0 286 323 relates to a two-step procedure for purifying polypeptides, especially factor VIII. The first step uses immobilized antibodies and the second step is based on an affinity region. The affinity region can be an ionic exchange gel or a HIC gel. Detergents are referred to primarily as active agents during the virus inactivation. They are also mentioned, in the prior art, to suppress the intermolecular association mediated by ionic forces, but there is no information about the use of detergents during the adsorption phase on the affinity region.
U.S. Pat. No. 4,743,680 also relates to buffer compositions during purification of factor VIII (anti-hemophilic factor or AHF), by column chromatography. Detergents are mentioned to promote elution from a mixed-function affinity chromatography gel, such as aminohexyl Sepharose.RTM.. There is no information in U.S. Pat. No. 4,743,680 about the use of detergents during the adsorption phase on a hydrophobic resin.
The difficulties met when applying HIC to factor VIII purification, can be attributed to the problems encountered when trying to establish a suitable retention window as well as elution conditions. A suitable retention window means that the factor VIII molecules are retained on the surface of the HIC gel by hydrophobic interaction, while other impurities, mainly nucleic acids and proteins are retained to a lesser degree or, preferably, not at all. A suitable retention window for factor VIII further means, that the factor VIII molecules can be eluted without too severe conditions, thus avoiding denaturation. Such a window can be obtained by selection of a suitable gel consisting of a matrix and hydrophobic ligands attached thereto. The type and density of the ligands strongly influence the interaction between the factor VIII molecules and the surface of the HIC gel, and thereby the retention window. Further parameters influencing the retention window are e.g. the ionic strength, temperature, pH and length of column.
It is of special importance for the selectivity of the process, that such non-specific adsorption factors as positive charges are avoided, since nucleic acids, which are negatively charged, are tolerated at an extremely low level in preparations of therapeutic proteins. Thus, in the purification of therapeutic proteins produced by a recombinant DNA technique, it is well known that considerable problems are encountered when trying to reduce the content of DNA to the very low level stipulated by the Food and Drug Administration (FDA) of the U.S.A recent example is disclosed in P. Ng and G. Mitra of Miles Inc. in the U.S., J. Chrom., A 658, p. 459 (1994), where the concentration of DNA was reduced but only to about 1 ng per dosage of a therapeutic protein as determined by the .sup.32 P R3 DNA hybridization method.
Pure hydrophobic interaction chromatography, using non-charged gels in combination with mild elution conditions, would provide another dimension for purifying recombinant factor VIII, since additional separation criteria would be utilized compared to those already used in ion-exchange and immunoaffinity chromatography. This would increase the number of process techniques available to optimize the overall purification of recombinant factor VIII. Furthermore, it would be valuable to find conditions for a moderately strong adsorption, permitting desorption by mild elution conditions, since a too strong adsorption might change the conformation of the protein.